T-cells belong to the lymphocytes and are responsible for a number of key functions in the immune system. In mammals, T-cells (thymocytes) differentiate in the thymus gland from hematopoietic progenitor cells formed in bone marrow. Part of the differentiation process is the expression of characteristic surface receptors, mainly the glycoproteins CD4 and CD8. T-cells expressing CD4, so-called CD4+ T-cells, bind MHC class II complexes (Reinerz and Schlossman, Cell 19, 821-827 (1980); Reinerz et al., PNAS USA 77, 1588-1592 (1980)), while CD8+ T-cells bind MHC class I complexes (Fitch, Microbiol. Rev. 50, 50-69 (1986)). T-cells are released into blood and lymph.
CD4 positive cells can differentiate into T helper subpopulations (Th1 and Th2), but also into regulatory T-cells. Regulatory T-cells can be further divided into subclasses, the thymus derived (nTreg) inducible ones (iTregs) being the most evaluated.
Although there are other Treg subpopulations, such as for example Tr1 or Th3, the present invention refers to CD4 positive thymus derived Tregs (nTregs) and inducible Tregs, both expressing the transcription factor Foxp3. As a major difference Foxp3 is stably and permanently expressed in nTregs confirming the irreversible Treg phenotype, whereas inducible Tregs display inducible or transient Foxp3 expression, which is reversible.
Tregs secrete immunomodulatory cytokines such as IL-10, TGF beta or IL-35 and exert suppressive activity on effector T-cells via several mechanisms, for example via suppression of the production of proinflammatory cytokines, direct cell-cell contact and modulating the activation state or function on antigen presenting cells (APC) (Shevach et al., Immunity (2009) 30; 636-645). A main characteristic of CD4 positive CD25 Treg cells is their anergic phenotype, meaning that they do not proliferate upon TCR stimulation, which can be restored by the addition of exogenous IL-2.
A prominent role for Tregs comprises maintaining homeostasis concerning immune responses and self tolerance. Treg dysfunction is correlated with autoimmune diseases.
Commonly, regulatory T-cells can be isolated via the surface receptor glycoproteins CD4, CD25, and characterized by intracellular staining of FOXP3. A further surface protein represents CD127 (IL-7 R), which is downregulated in Treg cells, and can be used for further purification of Tregs. Additionally, expression of CD39 (endonucleotidase) (Borselino et al., Blood (2007) 110, 1225-1232) or GARP (glycoprotein A repetitions predominant (GARP, or LRRC32) (Wang et al., PNAS (2009) 106, 32. 13439-13444).
Human CD4 is encoded on chromosome 12 and belongs to the immunoglobulin (Ig) superfamily. Its natural function as a T-cell surface receptor is related to T-cell activation by binding of MHC class II complexes. In addition, CD4 can bind the HIV-1 gp120 protein, the P4HB/CDI protein, and human herpes virus HHV-7 capsid proteins. Interactions with the HIV-1 gp120 and Vpu proteins also have been reported. CD4 has 458 amino acids. The peptide sequence is shown in FIG. 1.
The UniProt entry P01730 provides the domain structure of CD4 as shown below in Table 1, and in FIG. 6. The first 25 amino acids are a signal peptide, which is cleaved off in the biologically active form. Positions 26 through 396 constitute the extracellular domain, which is followed by the transmembrane region, positions 397 through 418. Asn296 and Asn325 are known glycosylation sites (König et al., J. Biol. Chem. 263, 9502-9507 (1988); Carr et al., J. Biol. Chem. 264, 21286-21295 (1989)).
The last part, positions 419 through 458, is the cytoplasmic domain. Here is the binding site for the Tyrosine protein kinase LCK (p56lck) (Rudd et al., PNAS USA 85, 5190-5194 (1988); Veillette et al., Cell 55, 301 (1988)), which is part of the signaling pathway activated by ligands binding to CD4.
TABLE 1Table showing the domain structure of CD4 (according to UniProt P01730)FeaturePositionsLengthDescriptionSignal peptide1-2525Chain26-458433T-cell surface glycoprotein CD4Topological domain26-396371ExtracellularTransmembrane region397-41822PotentialTopological domain419-45840Cytoplasmic (potential)Domain26-125100Ig-like V-typeDomain126-20378Ig-like C2-type 1Domain204-31778Ig-like C2-type 2Domain318-37478Ig-like C2-type 3Region427-45529HIV-1 Vpu-susceptibility domainGlycosylation site2961NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-2)Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAcGlycosylation site3251NeuAc(a2-3)Gal(b1-4)GlcNAc(b1-2)Man(a1-3)[Gal(b1-4)GlcNAc(b1-2)Man(a1-6)]Man(b1-4)GlcNAc(b1-4)GlcNAcDisulfide bond41 ←→ 109Disulfide bond155 ←→ 184Disulfide bond328 ←→ 370Lipidation site4191S-palmitoyl cysteineLipidation site4221S-palmitoyl cysteine
The extracellular part comprises 4 immunoglobulin-like domains. The first one, the N-terminal domain, comprising positions 26 through 125 is an Ig-like V-type domain. Based on the homology to antibodies, it has three homologues of antigen-complementary-determining regions, CDR1, CDR2, and CDR3 (Ashkenazi et al., PNAS USA 87, 7150-7154 (1990)) (see FIG. 6). The CDR1 and CDR2 spans are involved in the binding of class II MHC molecules (Moebius et al., PNAS USA 89, 12008-120012 (1992)), the gp120 HIV-1 envelope protein (Moebius et al., J. Exp. Med. 176, 507-517 (1992)) and anti-CD4 antibodies (Lanza et al., PNAS USA 90, 11683-11687 (1993)). Phe68 of CDR2 plays a key role for recognition and binding of class II MHC molecules and the gp120 HIV-1 envelope protein (Sharma et al., Biochemistry 44, 16192-16202 (2005)). All known ligands of CD4 bind to the N-terminal Ig-like V-type domain.
The mechanism of how regulatory T cells work is not fully clear. CD4+CD25+ Tregs inhibit polyclonal and antigen-specific T cell activation. The suppression can be mediated e.g. by a cell contact-dependent mechanism that requires activation of CD4+CD25+ Tregs via the TCR but Tregs do not show a proliferative response upon TCR activation or stimulation with mitogenic antibodies (anergic) (Shevach, Nature Rev. Immunol 2: 389 (2002). Once stimulated, they are competent to suppress in an antigen-independent manner the response of CD4+ T cells and CD8+ T cells as well as inhibit B-cell activation and clonal expansion.
The ability of CD4+CD25+ regulatory T cells to have a controlling influence on immune system activity has meant that they have been recognized as a potential target for treating diseases, such as autoimmune diseases, where it is desirable to exert a control on the immune system.
Autoimmunity is the failure of an organism to recognise its own constituent parts (down to sub-molecular levels) as “self”, which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Autoimmune diseases include multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, colitis ulcerosa, Crohn's disease, Type I Diabetes Mellitus (T1D), myasthenia gravis (MG), autoimmune polyglandular syndrome type II (APS-II), Hashimoto's thyroiditis (HT), systemic lupus erythematosus (SLE), Sjörgens Syndrome and autoimmune lymphoproliferative syndrome (ALS).
Autoimmune disease occurs when T cells recognise and react to ‘self’ molecules, that is, molecules produced by the cells of the host. Activation of ‘autoreactive’ T cells by presentation of autoantigens processed by antigen presenting cells (APC) leads to their clonal expansion and migration to the specific tissues, where they induce inflammation and tissue destruction.
Suppression of these T effector cell function by using immunosuppressive drugs is a principal therapeutic strategy that has been used successfully to treat autoimmune diseases. However these drugs induce general immune suppression due to their poor selectivity, resulting in inhibition of not only the harmful functions of the immune system, but also useful ones. As a consequence, several risks like infection, cancer and drug toxicity may occur.
It is generally agreed that CD4+ T cells play a major part in initiating and maintaining autoimmunity. Accordingly, it has been proposed to use mAbs against CD4+ T cells surface molecules, and in particular anti-CD4 mAbs, as immunosuppressive agents. Although numerous clinical studies confirmed the potential interest of this approach, they also raised several issues to be addressed in order to make anti-CD4 mAbs more suitable for use in routine clinical practice.
Several different mechanisms of action for CD4 mAbs have been proposed including: (1) antagonism of CD4-MHC II interactions resulting in inhibition of T cell activation, (2) CD4 receptor modulation as determined by a decrease in cell surface expression of CD4, (3) partial signaling through the CD4 receptor in the absence of T cell receptor cross-linking which can suppress subsequent T cell activation and trigger CD4 T cell apoptotic death, (4) Fc-mediated complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) leading to CD4 T cell depletion, and (5) stimulation of regulatory T cells.
Several anti-CD4 antibodies targeting T cells have been in clinical development (Schulze-Koops et al., J. Rheumatol. 25(11): 2065-76 (1998); Mason et al., J. Rheumatol. 29(2): 220-9 (2002); Choy et al., Rheumatology 39(10): 1139-46 (2000); Herzyk et al., Infect Immun. 69(2): 1032-43 (2001); Kon et al., Eur Respir J. 18(1): 45-52 (2001); Mourad et al., Transplantation 65(5): 632-41 (1998); Skov et al., Arch Dermatol. 139(11): 1433-9 (2003); Jabado et al., J. Immunol. 158(1): 94-103 (1997)) mainly aiming at CD4 cell depletion with only a few CD4 antibodies having been attributed to the other mechanisms like TRX-1, TNX-355, IDEC-151, OKTcdr4A.
The approach of using agents aimed at the activation of regulatory T cells for the therapy of autoimmune diseases has proven to be extremely difficult. Activation of Tregs via the TCR using the agonistic anti-CD3 antibody OKT-3 (Abramowicz et al, N Engl. J. Med. 1992 Sep. 3; 327(10):736) or via the co-stimulatory molecule CD28 using the superagonistic anti-CD28 antibody TGN 1412 lead to complete depletion of regulatory T cell population as well as other conventional T cells and the systemic induction and release of excessive amounts of pro-inflammatory cytokines including IFN-γ, TNF-α, IL-1 and IL-2, resulting in a clinically apparent cytokine release syndrome (CRS) in humans (Suntharalingam et al, N Engl. J. Med. 2006 Sep. 7; 355(10):1018-28).
However, recently humanized anti-CD4 antibodies have been described in WO2004/083247 which are capable of activating CD4+CD25+ regulatory T cells. The antibodies described in WO2004/083247 are humanized versions of the mouse antibody, mB-F5, a murine IgG1 anti-human CD4 described by Racadot et al. (Clin. Exp. Rheum., 10, 365-374 (1992)). The epitope of mB-F5 was reported by Racadot et al., as spanning the Ig-like C2 type 1 and type 2 domains of human CD4 from amino acid 162 to amino acid 232 as shown in FIG. 6.
Subsequent clinical trials reported in WO2009/112502, WO2009/121690, WO2009/124815 and in WO2010/034590, using one of these antibodies, designated BT061 (a humanized monoclonal IgG1), has resulted in the successful treatment of patients suffering from psoriasis and rheumatoid arthritis, providing proof that these antibodies are capable of treating autoimmune diseases safely and with good efficacy.
The promising clinical results achieved has increased the interest in providing further therapeutic agents having similar properties. It is therefore the aim of the present invention to provide screening methods for identifying such agents, and to provide further therapeutic agents.
Accordingly the present invention provides a method for screening for a molecule capable of binding to CD4 comprising:    (a) providing one or more candidate molecules;    (b) determining whether the one or more candidate molecules is capable of binding to one or more of the following regions of human CD4: amino acids 148 to 154, amino acids 164 to 168 and amino acids 185 to 192; and    (c) selecting a molecule determined in step (b) to be capable of binding to CD4.
The present inventors have unexpectedly found that the humanized antibody BT061 binds to a domain of CD4 which was previously unrecognized as a ligand binding site. This finding is particularly surprising given what was known in the art as the epitope for the murine antibody, mB-F5, from which BT061 was derived. The present inventors have also established the residues of BT061 that are involved in binding the CD4 molecule and have surprisingly found that not all of the CDRs of BT061 are involved in CD4 binding.
The identification of the binding region, and details of the mechanism of binding, has enabled the development of further screening methods, and of antibodies and antibody fragments capable of activating CD4+CD25+ regulatory T cells.
Accordingly, the present invention also provides a method for screening for an antibody or antibody fragment capable of binding with CD4 comprising:    (a) providing an antibody or antibody fragment comprising CDR1 and CDR2 of BT061 light chain and CDR1 and CDR3 of BT061 heavy chain optionally with amino acid substitutions in the sequences of the CDRs provided:    (i) the light chain CDR1 comprises: Ser32; Gly33; and Tyr 34;    (ii) the light chain CDR2 comprises: Leu54; and Ile57;    (iii) the heavy chain CDR1 comprises Asp31, Glu31, Thr31, Cys31, Pro31, Met31 or Tyr31; and    (iv) the heavy chain CDR3 comprises Tyr103, Phe103 or His103; Arg104; Tyr105; Asp106; and Trp110, Phe110, His 110 or Tyr110,    (b) determining whether the antibody or antibody fragment is capable of binding to CD4, and    (c) selecting the antibody or antibody fragment determined in step (b) to be capable of binding to CD4,    wherein the antibody or antibody fragment does not comprise CDR1, CDR2 and CDR3 of BT061 heavy chain and CDR1, CDR2 and CDR3 of BT061 light chain.
Still further the present invention provides an antibody or antibody fragment capable of activating CD4+CD25+ regulatory T cells comprising an antibody or antibody fragment capable of activating CD4+CD25+ regulatory T cells comprising CDR1 and CDR2 of BT061 light chain and CDR1 and CDR3 of BT061 heavy chain optionally with amino acid substitutions in the sequences of the CDRs provided:    (i) the light chain CDR1 comprises: Ser32; Gly33; and Tyr 34;    (ii) the light chain CDR2 comprises: Leu54; and Ile57;    (iii) the heavy chain CDR1 comprises Asp31, Glu31, Thr31, Cys31, Pro31, Met31 or Tyr31; and    (iv) the heavy chain CDR3 comprises Tyr103, Phe103 or His103; Arg104; Tyr105; Asp106; and Trp110, Phe110, His 110 or Tyr110,    and wherein the antibody or antibody fragment does not comprise CDR1, CDR2 and CDR3 of BT061 heavy chain and CDR1, CDR2 and CDR3 of BT061 light chain.